Beta-Oxidation of fatty acids in liver occurs in mitochondria as well as peroxisomes. As much as 25% of hepatic beta-oxidation occurs in peroxisomes. Considerable data indicate that flux of different chain length acylCoA thiolesters through mitochondrial beta-oxidation is regulated by the relative concentrations of three acyl CoA dehydrogenases as well as by chain-length dependent anticooperative binding of substrates to the general acyl CoA dehydrogenases (GAD). A major goal of this proposed research is to obtain structural evidence for that anticooperative behavior. A second major unsolved problem in the biochemistry of acyl CoA dehydrogenases is the nature of the extremely high affinity of the reduced dehydrogenases for their physiological oxidant, the electron transfer flavoprotein (ETF). Studies are proposed to elucidate the structural bases of nonindependent substrate binding by the GAD and interaction of reduced GAD with the ETF. These experiments will utilize techniques of protein modification, primary sequence determination and X-ray crystallography. The second major aim of the proposed research is to evaluate the physiological role and significance of peroxisomal fatty acid beta-oxidation in liver using animal models of disorders lipid metabolism including a model of diabetes and obesity. We also propose to determine whether heart, which utilizes fatty acid as a principal metabolic fuel, possesses a peroxisomal beta-oxidation system and whether that system proliferates in response to hypolipidemic drugs as does the hepatic system. Finally, we intend to investigate the properties of the peroxisomal acyl CoA oxidase and 3-ketoacyl CoA thiolase from rat liver since the enzymes may be key regulatory loci for peroxisomal beta-oxidation. Thus, investigations of the relative contribution of mitochondrial and peroxisomal beta-oxidation in models of perturbed lipid metabolism will expand our understanding of the role of peroxisomal fatty acid oxidation and the physiological chemistry of peroxisome proliferation. Investigations of key peroxisomal beta-oxidation enzymes will compliment studies at the organ level and will provide insight into reactions that may regulate flux through this extra-mitochondrial fatty acid oxidation system.